Coronaviruses are a medically significant group of enveloped, positive stranded RNA viruses that cause primarily respiratory and enteric infections in humans and many animals. The overall focus of this proposal is to understand the mechanism and function of coronavirus proteins in virus assembly. The virion envelope contains at least two major structural proteins, the membrane (M) and spike (S) proteins. Additionally, a few molecules of the envelope (E) protein are also present in the envelope. The nucleocapsid (N) phosphoprotein encapsidates the genomic RNA packaged inside the envelope. The N protein binds the genomic RNA to form a helical nucleocapsid. The most abundant structural protein in the virion envelope is the M protein. It interacts with itself, other structural proteins and the nucleocapsid and therefore plays a key role in viral assembly. The M protein contains a short N-terminal domain outside the virion, three transmembrane domains and a long C-terminus domain inside the virion. The C-terminus contains a long amphipathic domain and a highly charged hydrophilic tail. The amphipathic domain has been implicated to play a role in various viral protein-protein interactions, including M-S and M-E associations. Additionally, the extreme C-terminal -25 amino acids of M have been shown to be functionally important in assembly through interactions with the nucleocapsid. The specific residues and requirements that are involved in mediating these protein-protein interactions are not fully defined and understood. The specific aims of the proposed research are to determine the role of a conserved domain and charged residues within the M protein cytoplasmic tail that are important in coronavirus assembly. To achieve this goal, a panel of mutant viruses will be generated with various changes in charged residues and the conserved domain within the M tail. All of the mutants will be analyzed in the context of the full-length mouse hepatitis virus (MHV-CoV)-A59 infectious clone. Mutants will be studied for plaque morphology and growth kinetics compared to wild-type virus. Biochemical analysis, coimmunoprecipitation (co-IP) and confocal microscopy will be used to elucidate the mechanistic roles of M in assembly, in terms of specific residue and structure requirements. The proposed study will provide information which is applicable to identifying potential antiviral targets. Antiviral development against coronaviruses, including the recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV), will provide a significant improvement in health care treatment options. Antiviral reagents and vaccines directed towards coronaviruses will aid in the treatment and prevention of respiratory and enteric infections, thus contributing to global improvement of human health.